Micro-confinement of bacteria into w/o emulsion droplets for rapid detection and enumeration

Abstract Today, rapid detection and identification of bacteria in microbiological diagnosis is a major issue. Reference methods usually rely on growth of microorganisms, with the drawback of lengthy time-to-result. The method provides global information on a clonal population that is known to be inhomogeneous relative to metabolic states and activities. Therefore, there may be a significant advantage of methods that allow characterisation of individual bacteria from a large population, both for test time reduction and the clinical value of the characterisation. We report here a method for rapid detection and real-time monitoring of the metabolic activities of single bacteria. Water-in-oil emulsions were used to encapsulate single Escherichia coli cells into picolitre (pL)-sized microreactor droplets. The glucuronidase activity in each droplet was monitored using the fluorogenic reporter molecule MUG (4-methylumbelliferyl-β- d -glucuronide) coupled to time-lapse fluorescence imaging of the droplets. Such bacterial confinement provides several major advantages. (1) Enzymatic activities of a large number of single bacterium-containing droplet could be monitored simultaneously, allowing the full characterisation of metabolic heterogeneity in a clonal population. We monitored glucuronidase enzymatic activity and growth over ∼200 single bacteria over a 24-h period. (2) Micro-confinement of cells in small volumes allows rapid accumulation of the fluorescent metabolite, hence decreasing the detection time. Independent of the initial concentration of bacteria in the sample, detection of the presence of bacteria could be achieved in less than 2 h. (3) Considering the random distribution of bacteria in droplets, this method allowed rapid and reliable enumeration of bacteria in the initial sample. Overall, the results of this study showed that confinement of bacterial cells increased the effective concentration of fluorescent metabolites leading to rapid (2 h) detection of the fluorescent metabolites, thus significantly reducing time to numeration.

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